EKOLOGIJA. 2008. Vol. 54. No. 1. P. 40–43DOI: 10.2478/V10055-008-0008-y© Lietuvos mokslų akademija, 2008© Lietuvos mokslų akademijos leidykla, 2008

Evidence for a risk of tick-borne infection in the city parks of Vilnius, Lithuania

Th irty-six adult ticks collected in the parks of the Lithuanian capital Vilnius in the spring of 2005 were identifi ed as Ixodes ricinus (L.) and studied for the presence of live pathogens by darkfi eld microscopy, of exoskeleton anomalies by light stereomicroscopy, and of several tick-borne pathogens by PCR. One of the ticks was positive for three pathogens simultaneously: Ehrlichia muris (monocitic ehrlichiosis agent), Borrelia afzelii, and Borrelia garinii; one was du-ally infected by both species of Borrelia. In addition, ticks positive for several species of Borrelia were detected: B. afzelii (n = 5), B. garinii (n = 2), some other species of B. burgdorferi sensu lato (n = 2), Ehrlichia muris (n = 1). Th e total proportion of infected ticks was 27.8% (10 of 36), of which 20% (2 of 10) were multiply infected; 47.2% of ticks had exoskeleton anomalies, and such specimens had more multiple infections (35.3% versus 21%). One triply infected tick was found only among the anomalous specimens. Live spirochaetes prevailed among the anomalous specimens: 35.3% (6/17) vs. 5.3% (1/19). Th e diff erence was statistically signifi cant: chi square was equal 5.166, P < 0.05. In conclusion, even this small sample size reveals several important tick-borne pathogens in the newly detected focus in the centre of Vilnius, transmitted by I. rici-nus. It suggests that people visiting these parks are exposed to the risk of tick-borne infection, especially when resting on the grass.

Key words: park, tick Ixodes ricinus, exoskeleton anomalies, tick-borne infection prevalence

Milda Žygutienė 1,

Andrey Alekseev 2,

Helen Dubinina 2,

Rita Kazlauskienė 3

1 Centre for Communicable Diseases Prevention and Control, LT-08221 Vilnius, Lithuania E-mail: eugezygu@takas.lt

2 Zoological Institute, Russian Academy of Sciences, 199034 St.-Petersburg, Russia E-mail: devana@hd1389.spb.edu

3 National Public Health Investigations Centre, LT-10210 Vilnius, Lithuania

INTRODUCTION

Ticks of the genus Ixodes, which have a circumpolar distribu-tion within the forested areas of the Northern hemisphere, are well known as vectors of bacteria belonging to the groups of Spirochetales (genus Borrelia), Rickettsiales (genera Ehrlichia and Anaplasma), piroplasmids (protists of the genus Babesia) and fl aviviruses. However, in Western Europe the main vector for tick-borne pathogens, Ixodes ricinus (L.) has become com-mon not only in wild-forested areas, but also in green patches near and inside towns and especially in their park areas. It is necessary to stress that in the Baltic countries Latvia and Estonia and in North-western Russia there are large areas where I. rici-nus and Ixodes persulcatus Schulze coexist (Ecker et al., 1999; Haglund et al., 2003; Charrel et al., 2004; Golovljova et al., 2004) and, parasitizing on similar hosts, are able to exchange various pathogens. In town parks, especially in those distributed in the centre or near a city, favourable conditions exist for I. ricinus ticks. Th ese ticks are very oft en infected with tick-borne patho-

* Corresponding author: Zoological Institute RAS, Universitetskaya nab., 1, 199034, St. Petersburg, Russia.

gens and can thus be dangerous for the park’s visitors, especially when resting on the grass.

In a recreational park in south-western Ireland, I. ricinus ticks infected by Borrelia burgdorferi sensu lato were numer-ous on the edges of paths and roads. Gray et al. (1999), who de-scribed this phenomenon, concluded that the main source of infection for ticks was not rodents, but birds. Bašta et al. (1999) hold a similar opinion, because in the Prague (Czech Republic) urban parks of Petřin, Vitkov and Stromovka where ticks were regularly collected, the authors found mainly B. garinii which is transported by birds, and fewer B. afzelii associated with rodents. Th e infection rate was high; every tenth tick was infected.

In another study, I. ricinus ticks were collected in a green sub-urban area of Chisinau, Moldova, in the spring of 2005 by Koči et al. (2007) who revealed not only 5 species of B. burgdorferi s.l. (again mainly (42%) B. garinii, commonly found in birds), but also Anaplasma phagocytophilum, an agent of a disease in which human blood granulocytes are aff ected.

Alekseev et al. (2003) worked in a suburb forested recreation zone 28 km from the centre of St. Petersburg. Th is study revealed 7 diff erent pathogens of viral, bacterial and protozoan origin (maxi-mum 3 simultaneously in one I. persulcatus female). Th ese authors

41Evidence for a risk of tick-borne infection in the city parks of Vilnius, Lithuania

found one I. persulcatus female quickly moving in the grass, pro-viding evidence for the possibility of exposure of humans to ticks in urban parks. Even on comfortable benches near green grass-plots, it is necessary to be careful. Th rushes, starlings and other birds can also spread ticks (Alekseev et al., 2001).

Pollution can also aff ect the spread of disease, and cities are commonly polluted with heavy metal ions, cadmium among them, from industrial and automobile exhaust. As proven previ-ously (Alekseev, Dubinina, 1996; Dubinina et al., 2004; Alekseev et al., 2007), ticks in polluted areas become more dangerous as vectors and have some exoskeleton deformations which may contribute to their identifi cation.

Th e main source of cadmium for rodents, granivorous birds, hosts of tick larvae and nymphs, is vegetation which can arise from polluted soil. Within the last few years, the literature re-garding the infl uence of heavy metals on the growth, develop-ment and size of diff erent kinds of vegetation is steadily increas-ing (Гладков, 2007).

Th e above data prompted us to investigate ticks collected in Vilnius parks for the presence of tick-borne pathogens and of exoskeleton anomalies. Furthermore, the purpose was to con-fi rm whether the possible anomalies can be linked to multiple infections of I. ricinus whatever the region.

MATERIALS AND METHODS

Ticks. Adults and nymphs of I. ricinus were collected by fl agging in the Lithuanian capital Vilnius parks (Vingis and Bukčiai) lo-cated in the central part of the city, in 2005, from April 15 till May 02. Th e total fortnightly yield of collected ticks was not great: 62 specimens were collected, among them 23 nymphs (37%) and 39 adults. Only adults were investigated for the pres-ence of anomalies and pathogens. Exoskeleton anomalies were revealed by light stereomicroscopy.

Pathogen detection methods. Th e hind part of adult specimens was dissected, and live spirochaetes were observed and calculated in 200x microscope dark fi elds in a drop of saline solution.

Adults, fi xed in 70% ethanol, were transferred to St. Petersburg to observe specimens with anomalies of their exo-skeletons and then to investigate them using PCR with species-specifi c primers to identify whether they were infected by

B. afzelii, B. garinii, E. muris, A. phagocytophilum or Babesia microti. Tick-borne encephalitis was not investigated in this study.

Preparation of ticks for pathogen identifi cation. Adult ticks were rinsed in 70% alcohol, then dried, frozen in liquid nitrogen and crushed individually. To every sample, 200 μl sterile H2O, 3 μl transfer-RNA solution and 200 μl 4 M guanidine thiocyanate were added, followed by 10 min of incubation at 0 °C. Aft er incubation, 200 μl of phenol : chloroform : isoamyl alcohol (25 : 24 : 1) solu-tion was added. Samples were frozen at –30 °C and then centri-fuged for 4–5 min at 10,000× g. Th e supernatant was transferred into a polypropylene tube and mixed with 1/10 vol. of 2 M sodium acetate (pH 4.5) and an equal volume of isopropyl alcohol. Th e samples were frozen in liquid nitrogen, then thawed, and cen-trifuged for 10 min at 20,000× g. Th e supernatant was removed. RNA/DNA pellets were washed with 0.5 ml of 75% ethanol, dried and redissolved in 150 μl H2O. Samples were used for PCR either immediately or aft er storage at –70 °C.

PCR assay. Polymerase chain reactions (PCR) were per-formed in a volume of 25 μl using a PerkinElmer 9600 ther-mal cycler. Each PCR mix contained 200 μM each of dNTP, 2 U Taq polymerase, 0.5 μM of each primer and 10 μl of template DNA. Th e primers are listed in Table 1. Th e primers for Borrelia species, A. phagocytophilum and E. muris / E. chaff eensis were supplied by DNA Technology (original method of “Helix” en-terprise, St. Petersburg, Russia). For Babesia microti / Babesia odocolei and for TBEV detection, the primers were from ASB “Vector-Best” (Novosibirsk, Russia). dNTP, Taq-polymerase, RT-set, and buff ers were from “DNA-Technology” (Russia) and MBI “Fermentas” (Lithuania).

For B. burgdorferi s.l. a three-step cycling program was used: an initial 1-min denaturation at 94 °C, 35 cycles of 30-s dena-turation at 94 °C, 40-s annealing at 59 °C, 40-s polymerization at 72 °C, and a fi nal 3-min extension at 72 °C. Identical proce-dures were used for the other Borrelia species, but with anneal-ing temperatures of 51 °C for B. burgdorferi sensu stricto, 48°C for B. afzelii, and 50 °C for B. garinii. Th e PCR cycles for other microbes were as follows:

for Ehrlichia sp. – an initial 1-min denaturation at 94 °C, 35 cycles of 30-s denaturation at 94 °C, 40-s annealing at 52 °C, 40-s polymerization at 72 °C, and 3-min extension at 72 °C;

Table 1. List of primersPrimer pairs Nucleotide sequence Amplicon size (bp) Target species/gene

F 5’-ATG TTG AAA TCT CAA GCT ATG AAG-3' 501 Borrelia burgdorferi R 5’-CTG TAG GCT ATT TTG AAT TGC AAG-3' s.l./16S RNA:F 5’-CCT ACA TCA ACC TTA AGT TGC T-3' 293 Borrelia R 5’-GCC AAG AGA AAT TGT TGT AAA TC-3' afzelii/16S RNAF 5’-CAA GCT CAG CTG CTG ATG CA-3' 287 Borrelia R 5’-GCC AAG AGA AAT TGT TGT AAA TC-3' garini/16S RNAF 5’-ACA ATT TCA AGC ACC ACT GAA-3' 286 Borrelia burgdorferi R 5’-GCC AAG AGA AAT TGT TGT AAA TC-3' s.s./16S RNAF 5’-AGG AAG CGT AAT GAT GTC TAT GG-3' 553 Anaplasma phagocyto-R 5’-TCC CAT CGA TAC TAG GGT AAG AGA-3' philum/16S RNA:F 5’-ATC TGT TTA TTA TTT GCA GCA A-3' 587 Ehrlichia muris Ehrlichia R 5’-GAG ATA TTG TTT TAT TAT AGA TAG-3' chaff eensis/16S RNA

PIRO-A 5’-AAT ACC CAA TCC TGA CAC AGG G-3' 438/408 Babesia microti Babesia odocolei/ PIRO-B. 5’-TTA AAT ACG AAT GCC CCC AAC-3' nss-rDNA

42 Milda Žygutienė, Andrey Alekseev, Helen Dubinina, Rita Kazlauskienė

for Babesia sp. – an initial 1-min denaturation at 94 °C, 35 cycles of 30-s denaturation at 94 °C, 40-s annealing at 60 °C, 40-s polymerization at 72 °C, and 3-min extension at 72 °C.

All samples that were PCR-positive with Bab-1 and Bab-4 primers, indicating the presence of Babesi sp., were tested with PIRO-A and PIRO-B primers; 438-bp-sized fragments were vis-ualized in electrophoresis. Babesia microti can be distinguished from Babesia odocolei, Babesia divergens and Babesia capreoli by the PCR restriction fragment length polymorphism (RFLP) as-say. Babesia microti is characterized by digestion of the 438-bp amplicon by the Hinf I enzyme producing two fragments, 356 and 81 bp in length, and by resistance to digestion by Bst E II (Armstrong et al., 1998; Duh et al., 2001).

Statistical methods. Pearson’s chi-square test (STATISTICA for Windows 6) was used to estimate the signifi cance of diff er-ences between infection prevalence in anomalous and normal ticks. A p value of 0.05 or less was considered signifi cant.

RESULTS

Th irty-six specimens were studied for the presence of exoskel-eton anomalies and infestation by live spirochaetes. In 17 of 36 ticks (47.2%) anomalies were revealed. Among them (Table 2), 6 specimens contained spirochaetes (35.3%), whereas among 19 normal specimens only 1 (5.3%) was infested by this micro-organism (the chi-square value between the absolute values of fi gures 5.166, P < 0.05). Among 7 positive ticks, 6 were males and only 1 was female. Th e number of spirochaetes only once exceeded 10 (in one male it was 28).

Th ere were no coincidences between the detection of live spirochaetes and the presence in the same specimens of patho-genic borreliae or other agents. Th e latter were never discovered in the ticks in which live spirochaetes were revealed by darkfi eld microscopy.

Neither B. burgdorferi s.s. nor A. phagocytophlum or Bab. mi-croti were found in the small group of ticks studied. Nevertheless, 10 of 36 ticks contained microorganisms pathogenic to man. Th us, nearly every third tick was protentially dangerous. Among this lot of ticks, B. burgdorferi s.l. was found only twice: once as a single infection and one time together with E. muris. It is not possible to exclude that at least one of these species could be in-fected by Borrelia lusitaniae whose pathogenicity and distribu-tion has been observed in Europe but was not studied here. PCR analysis revealed B. afzelii in 7 ticks (5 times as a monoinfection and 2 in mixed ones), B. garinii was detected 4 times and in 2 of them together with other pathogens. Two times the agent of hu-man monocytic ehrlichiosis, E. muris, was revealed, once alone

and another time together with both mentioned above species of borreliae. Not only spirochaetes, but also agents pathogenic for man were more common among anomalous ticks.

Th e diff erence between the prevalence of spirochaetes in anomalous ticks versus normal ones was 2.8 times greater, whereas the prevalence of tick-borne pathogens in anomalous ticks versus normal specimens was only 1.8 times, but, never-theless, it was visible. Triple-infected ticks were observed only among the anomalous group of ticks (E. muris, B. afzelii and B. garinii). A comparison of diff erences using the chi-square in-dex concluded that the diff erence in both cases was statistically signifi cant (chi-square 5.055 and 5.166, P in both cases <0.05).

DISCUSSION

Even with a small sample size, we can conclude that infected ticks I. ricinus are common in the centres of small and large cit-ies. In addition, pollution might be greater inside urban terri-tories than outside of them. For example, on the Curonian Spit, part of which belongs to Russia and another part to Lithuania, in the national park territory, the number of anomalous tick was 42.5% in 2005, whereas in Vilnius parks in the same year and the same season of collection its number was somewhat higher (47.2%) (Table 2). Like in other green patches (for example, in Chishinau), the same two most common Borrelia species were found, but it was quite possible that by using other primers more representatives of the B. burgdorferi s.l. group could be revealed. In both places, human ehrlichiosis agents were found, but in Moldova the human granulocytic ehrlichiosis agent occurred both alone and as a mixed infection, whereas in Vilnius only the human monocytic ehrlichiosis agent E. muris was detected. In the St. Petersburg suburb forested recreation zone, among 147 infected I. persulcatus ticks (1.412 specimens were studied) one third of them had multiple infections. In Vilnius, 2 out of 10 (20%) of collected ticks were either dually or triply infected. Th us, the same trend is apparent despite the very small number of ticks studied.

Interestingly, in Finland on the Kokkola archipelago (Alekseev et al., 2007) which is far from any capitals or towns 40% (12 of 30) of ticks, had exoskeleton anomalies. Th ere, the more commonly found I. persulcatus contained tick-borne pathogens in 63.3% of cases (19 out of 30, and 12 were multiply infected). Th is diff erence is understandable since in the wild the sources of reservoirs of tick-borne pathogens are greater than in towns or their suburbs. Nevertheless, the described phenomena have more similarities than diff erences.

Table 2. Prevalence of live spirochaetes and other tick-borne agents in anomalous and normal Ixodes ricinus ticks collected in the city parks of VilniusTicks Microorganisms and No. of the number of observations

Characteristic No.Probable apathogenic

Pathogenic for manMonoinfection Dual Triple Prevalence

abs. % Ba Bg Em Ba + Bg Ba + Bg + Em abs. %Anomalous 17 6/17 35.3 3 2 0 0 1 6/17 35.3

Normal 19 1/19 5.3 2 0 1 1 0 4/19 21.0Chi-square 5.166 Chi-square between ratios 5.055

p <0.05 p <0.05

Ba – Borrelia afzelii, Bg – Borrelia garinii, Em – Ehrlichia muris.

43Evidence for a risk of tick-borne infection in the city parks of Vilnius, Lithuania

In conclusion, even this small sample size reveals several im-portant tick-borne pathogens in the centre of Vilnius, suggesting that people visiting these parks are exposed to the risk of tick-borne infections transmitted by I. ricinus, especially while rest-ing on the grass.

ACKNOWLEDGEMENTS

We wish to thank Dr. Vladimir Kulikov, the “Helix” manager, for valuable assistance and help in pathogen identifi cation, and both anonymous reviewers for valuable comments and advice.

Received 17 September 2007Accepted 20 December 2007

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Milda Žygutienė, Andrey Alekseev, Helen Dubinina, Rita Kazlauskienė

UŽSIKRĖTIMO ERKIŲ PLATINAMOMIS LIGOMIS RIZIKOS ĮRODYMAS VILNIAUS MIESTO PARKUOSE

S a n t r a u k aIštirtos trisdešimt šešios Ixodes ricinus (L.) erkės, 2005 m. pavasarį surinktos dviejuose Vilniaus parkuose dėl užsikrėtimo spirochetomis, egzoskeleto anomalijų ir keletos erkių platinamų ligų sukėlėjų. Vienoje iš erkių aptikta trys sukėlėjai: Ehrlichia muris (monocitinės erlichiazės sukėlėjas), Borrelia afzelii ir Borrelia garinii. Be to, tirtose erkėse nu-statyta B. afzelii (n = 5), B. garinii (n = 2), kiti B. burgdorferi sensu lato grupės genotipai (n = 2), Ehrlichia muris (n = 1). Iš viso rasta 27,8% (10/36) infekuotų erkių, dviejose iš jų (20%) nustatyta daugiau nei vie-nas ligų sukėlėjas; 47,2% erkių turėjo egzoskeleto anomalijas ir tokios erkės buvo labiau infekuotos (35,3 prieš 21%) ligų sukėlėjais. Triguba infekcija nustatyta tik tarp anomalių erkių. Gyvos spirochetos taip pat dažniau aptiktos anomaliose erkėse: 35,3 (6/17) prieš 5,3% (1/19). Rezultatai statistiškai patikimi. Nedidelės apimties tyrimai patvirtina, kad I. ricinus erkės, rastos Vilniaus parkuose, yra kelių užkrečiamųjų ligų sukėlėjos ir tai yra rizikos veiksnys parkų lankytojams.

Raktažodžiai: parkas, Ixodes ricinus, egzoskeleto anomalijos, erkių platinamų patogenų paplitimas